Inhibition of oxygen exchange by chemical modifiers at the sulfhydryl 1 or reactive lysine residue of myosin. Changing the rates of intermediate enzymatic reactions by modifying the course of reaction-linked conformational changes.
During the hydrolysis of MgATP by myosin, there is an extensive exchange of oxygen between water and the terminal phosphate group of bound nucleotide, which results from a repeated cycle of hydrolytic cleavage and its reversal. An analysis of the distribution of [18O]Pi species from the hydrolysis of [gamma-18O]ATP gives an estimate for the apparent rate constant [k-3(app)] of reverse cleavage, which is the rate-limiting step of the exchange cycle. We have performed this kind of analysis with two different forms of modified myosin, containing either N-ethylmaleimide at the sulfhydryl 1 group or trinitrophenyl at the reactive lysine residue. Although these modifications of the protein are chemically different, and the sulfhydryl 1 group and the reactive lysine residue are far apart in the primary chain of the myosin head, the two modifications caused a similar marked inhibition of oxygen exchange. This effect resulted from: 1) a decrease in the time available for the exchange cycle due to a 3-10-fold increase in the turnover rate of hydrolysis and 2) a reduction in k-3 (app) from 2-5 s-1 to 0.2-0.5 s-1. It is proposed that modifications of this type influence enzymatic activity by altering the course of "reaction-linked conformational changes," i.e. those changes in protein structure that are coupled to the catalytic mechanism but are not part of the substrate-binding site proper. It is also suggested that certain naturally occurring protein modifications, e.g. methylation of histidine or lysine residues, could regulate function by operation of this mechanism.
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D055598:Chemical Phenomena; D002621:Chemistry; D005033:Ethylmaleimide / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism*; D008433:Mathematics; D008958:Models, Molecular; D009218:Myosins / Q000378:metabolism*; D010100:Oxygen / Q000378:metabolism*; D010710:Phosphates / Q000378:metabolism; D011487:Protein Conformation; D011817:Rabbits
D010710:Phosphates; D000255:Adenosine Triphosphate; D009218:Myosins; D008239:Lysine; D005033:Ethylmaleimide; D010100:Oxygen